Osteoarthritis (OA) is a highly prevalent degenerative joint disease, which develops very slowly. Hallmarks of OA are destruction of the joint cartilage (loss of proteoglycans and collagens from the cartilage matrix), accompanied with damage to the underlying bone formation of osteophytes (bony projections along joint margins) and changes in the synovial membrane, leading to joint inflammation (synovitis). Altogether, these processes cause pain, misalignment and loss of function of the affected joints. Osteoarthritis is characterized by loss of joint cartilage that leads to pain and loss of function, typically in the knees and hips, and affects 81.4 million individuals in seven major markets, which are the US, Japan, France, Germany, Italy and Spain (Datamonitor Report DMHC2493, December 2009). In the US, the Center for Disease Control and Prevention (CDC) reports that, overall OA affects 13.9% of adults aged 25 and older and 33.6% (12.4 million) of those 65+(an estimated 26.9 million US adults in 2005 up from 21 million in 1990). Increases in life expectancy and ageing populations are expected to make osteoarthritis the fourth leading cause of disability by the year 2020 (Anthony D. Woolf & Bruce Pfleger, Burden of major musculoskeletal conditions, Bulletin of the World Health Organization 2003; 81:646-656). From the medical perspective, OA is also associated with other chronic disorders such as over-weight, diabetes, hypertension, dyslipidemia and coronary artery disease, which account for the multi-morbidity of subjects diagnosed with OA.
Currently, at the exclusion of replacement surgery, there is no cure for OA and available treatments aim at relieving symptoms and improving function. These include a combination of patient education, physical therapy, weight control, and use of medications (nutritional supplements, simple analgesics, topical NSAIDS, oral NSAIDS, cox-2-inhibitors, and oral steroids, intra-articularly administered steroids and hyaluronic acid).
In the past several attempts were made to inhibit the activity of those enzymes mainly involved in the degradation of the cartilage. Unfortunately with little success so far. Anti protease treatments lacked the required potency and clinical benefit (Lewis E J, Bishop J, Bottomley K M, Bradshaw D, Brewster M, Broadhurst M J, et al. Ro 32-3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo. Br J Pharmacol 1997; 121:540-6; Brown P D. Ongoing trials with matrix metalloproteinase inhibitors. Expert Opin Investig Drugs 2000; 9:2167-77), were not sufficiently bioavailable or orally active and/or lacked specificity to the targeted enzymes. Specifically, MMP inhibitors showed dose- and duration-dependent muskuloskeletal side effects consisting of joint stiffness, joint fibroplasias and accumulation of collagen type I in the osteoarthritic joints (Renkiewicz R, Qiu L, Lesch C, Sun X, Devalaraja R, Cody T, et al. Broad-spectrum matrix metalloproteinase inhibitor marimastat-induced musculoskeletal side effects in rats. Arthritis Rheum 2003; 48:1742-9; Rao B G. Recent developments in the design of specific Matrix Metalloproteinase inhibitors aided by structural and computational studies. Curr Pharm Des 2005; 11:295-322). With the increasing number of patients suffering from the disease, novel therapeutics are highly desirable and much sought after which are less invasive, do not only relieve the symptoms but improve the underlying cause of the disease or injury, i.e. the degradation of the cartilage.
With the increasing number of patients suffering from the disease, novel therapeutics are highly desirable and much sought after which are less invasive, do not only relieve the symptoms but improve the underlying cause of the disease or injury, i.e. the degradation of the cartilage.
The therapeutic protein CD-RAP was shown to be able to slow the degradation of cartilage as well as to improve the regeneration of damaged cartilage. However, in order to serve as source for a sufficient drug supply, CD-RAP also needs to meet the commercial requirements of high yield in production. Thus, it is desirable to produce CD-RAP by recombiant DNA-technology. A recombinant bacterial process could solve the problem by expressing the gene encoding CD-RAP under control of a strong bacterial promoter. Bacterial direct expression requires a Methionin at position −1 for initiation of translation. The codon has to be added to the sequence encoding CD-RAP sequence. The native CD-RAP contains at Position 3 of its sequence a Methionin. During expression this Methionin could be also used for translational initiation. Thus two expression products would be observed, one starting with Glycin to give rise to native CD-RAP and one starting with Proline to give rise to a truncated form of CD-RAP. Analysis of hitherto available CD-RAP preparations revealed that these preparations, in fact, contain at least three different CD-RAP species. In particular, the amino terminus of CD-RAP, e.g. expressed in E. coli, is sensitive to degradation, resulting in an about 10% N-terminal heterogeneity of the CD-RAP product. CD-RAP obtained by expression in E. coli contains at least the following three different protein species:
a) CD-RAP (108AA) having an additional N-terminal methionine, i.e. in total 108 amino acids, which is the major species (?. 90%) and has the N-terminus: MGPMPKL . . .
b) CD-RAP (107AA) consisting of the mature CD-RAP sequence without an additional N-terminal methionine and having the N-terminus GPMPKL . . . as well as
c) CD-RAP (105AA) being an N-terminal truncated CD-RAP having the N-terminal sequence: MPKL . . . , i.e. two amino acids less than the mature CD-RAP sequence.
This heterogeneity in product is not desirable as further purification steps lowering yield would become necessary. The heterogeneity could become even higher if the F-methionin would not be quantitatively removed posttranslationally. Furthermore, native CD-RAP exhibits a high hydrophobicity, which results in a restricted expression in bacteria due it its relative toxicity for the cells.
The recombinant expression of CD-RAP further requires the folding of the protein subsequent to its expression. However, folding yields of native CD-RAP were found to be very low, partially due to the high hydrophobicity.
To overcome these problems in the production of CD-RAP, a fusion protein of CD-RAP with a pre-sequence was designed, which leads to high yield in expression of this precursor CD-RAP protein, which may, amongst others, be due to the reduced hydrophobicity of the precursor protein. Moreover, the folding of the fusion protein was found to be much easier than that of the native CD-RAP. To constitute the native CD-RAP after the folding it is however necessary to remove the pre-sequence by an enzymatic cleavage. To achieve this, the interface between the amino acid sequence of the native CD-RAP and the pre-sequence was designed as cleavage site, in particular as cleavage site for an endoprotease. Overall, the productivity of CD-RAP is increased after its expression with a pre-sequence and the subsequent removal of the pre-sequence, up to 60 fold compared to expression without pre-sequence. Preparation of CD-RAP using the CD-RAP precursor protein disclosed herein allows for the provision of a CD-RAP preparation having a defined length of 107 amino acids and having a defined N-terminus. This is an essential advantage in addition to improved yield at the manufacture of CD-RAP.
To specifically target effected joints and decrease possible (e.g. systemic) side effects, there is also a need for formulations allowing for intra-articular administration of CD-RAP.
In this context, formulations are desired which are suitable for intravenous and intra-articular administration. Desirably, these formulations comprise excipients and buffer combinations that leads to increased stabilisation of the CD-RAP protein allowing for longer storage of the formulation. In particular, formulations are desired which allow CD-RAP to remain at the effected side for a longer period of time and to reach chondrocytes more effectively. To allow therapeutic and/or preventive administration such formulation needs to be sterilisable as well as exhibit e long shelf-life allowing for its storage. Therefore, a liposomal formulation of CD-RAP was developed which is suitable for intra-articular administration. A scalable process was developed to produce empty liposomes small enough to be sterilized by filtration. Formulation development studies allowed identifying the parameters influencing CD-RAP encapsulation efficiency in the liposomal product, which were found to be the buffer type, and the lipid content, as well as the reconstitution method for a freeze-dried formulation.
The general process of producing liposomal formulations involves the following steps used are: —Preparation of empty liposomes (small unilamellar vesicles, SUVs) by preparing a lipid blend which is hydrated and homogenised, and liposomes are then extruded; —Mixing of SUV suspension with CD-RAP aqueous solution, —Freeze-drying, —Reconstitution with water for injection (WFI).
At the end of this process, the polydispersity of the liposomes is rather large (about 0.3), which leads to sterile filtration problems. In order to simplify the process and reduce polydispersity, a solvent injection method was developed to prepare the empty liposomes. In this method there is no need to produce a lipid blend, thereby avoiding the preparation of an intermediate product and eliminating a process step. Solvent injection is a scalable method which allows for a straightforward scale up as well as avoiding sizing steps such as homogenisation and extrusion.